SPECIFIC SYNTHETIC CHIMERIC XENONUCLEIC ACID GUIDE RNA; s(XNA-gRNA) FOR ENHANCING CRISPR MEDIATED GENOME EDITING EFFICIENCY

ABSTRACT

The invention provides specific synthetic chimeric xenonucleic acid guide RNA; s(XNA-gRNA) for enhancing crispr mediated genome editing efficiency. The invention also provides methods and compositions for inducing CRISPR/Cas-based gene editing/regulation (e.g., genome editing or gene expression) of a target nucleic acid (e.g., target DNA or target RNA) in a cell. The methods include using single guide RNAs (sgRNAs) that have been chemically modified with xeno nucleic acids which enhance gene regulation of the target nucleic acid in a primary cell for use in ex vivo therapy or in a cell in a subject for use in in vivo therapy. Additionally, provided herein are methods for preventing or treating a genetic disease in a subject by administering a sufficient amount of a sgRNA that has been chemically modified with xeno nucleic acids to correct a mutation in a target gene associated with the genetic disease.

This application claims the priority benefit under 35 U.S.C. section 119of U.S. Provisional Patent Application No. 62/376,206 entitled “SpecificSynthetic Chimeric Xenonucleic Acid Guide RNA; s(XNA-gRNA) For EnhancingCRISPR Mediated Genome Editing Efficiency” filed on Aug. 17, 2016; andProvisional Patent Application No. 62/376,287 entitled “Synthetic RoutesTo Xenonucleic Acid (Xna) Monomers” which are in their entirety hereinincorporated by reference.

FIELD OF THE INVENTION

The present invention relates to the field of molecular biology. Inparticular, the present invention relates to the clusters of regularlyinterspaced short palindromic repeats (CRISPR) technology. Thisinvention also pertains to modified compositions for use in CRISPRsystems, and their methods of use.

The invention further relates to CRISPR-related methods and componentsfor editing of, or delivery of a payload to, a target nucleic acidsequence. The present disclosure also generally relates to compositionsand methods for the genetic modification of cells. In particular, thedisclosure relates to CRISPR reagents and the use of such reagents.

BACKGROUND OF THE INVENTION

Genome engineering can refer to altering the genome by inserting,deleting, mutating, or substituting specific nucleic acid sequences. Thealtering can be gene or location specific. Genome engineering can usenucleases to cut a nucleic acid thereby generating a site for thealteration. Engineering of non-genomic nucleic acid is alsocontemplated. A protein containing a nuclease domain can bind and cleavea target nucleic acid by forming a complex with a nucleic acid-targetingnucleic acid. In one example, the cleavage can introduce double strandedbreaks in the target nucleic acid. A nucleic acid can be repaired e.g.by endogenous non-homologous end joining (NHEJ) machinery. In a furtherexample, a piece of nucleic acid can be inserted. Modifications ofnucleic acid-targeting nucleic acids and site-directed polypeptides canintroduce new functions to be used for genome engineering.

After the human genome project, the sequence of every gene in the humangenome is now known. However, the function of most genes is still notclear. One of the most common strategies for studying the function of aparticular gene is to knock it out in a model organism. However, untilrecently, gene knockout could only be done in certain animals, and thecost has been very high. The CRISPR-Cas9 system has now emerged as apowerful genome-editing technology that can knock out any gene incultured cells with ease.

The use of clustered regularly interspaced short palindromic repeats(CRISPR) and associated Cas proteins (CRISPR-Cas system) forsite-specific DNA cleavage has shown great potential for a number ofbiological applications.

This powerful and revolutionary technology has already become one of themost commonly used tools in biological research. Almost all of the majorcompanies, including Life Technologies, Sigma, and Santa CruzBiotechnology, provide services based on this technology. Moreover,CRISPR-Cas9 technology has been actively pursued as a therapeutic toolfor treating various diseases. A CRISPR-Cas9 system with increasedknockout efficiency will be of great interest. The current commonly usedsingle-guide RNA (sgRNA) has a shortened duplex structure compared withthe native bacterial crRNA-tracrRNA duplex.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is amethod of gene editing that utilizes the Cas9 protein and specific guideRNAs to either disrupt host genes or insert sequences of interest.Initially used in bacteria as an adaptive immunity response, CRISPR hasbeen since utilized in the biological field as a new alternative togenome engineering. Furthermore, CRISPR provides a cheaper alternativeto other gene editing techniques such as zinc fingers, and is quicklybeing adopted as the technique of choice.

Cas9 (CRISPR associated protein 9) is a naturally occurring enzyme foundin some bacteria that is used for immunity. Cas9 works by using guideRNA with short sequences complimentary to potential foreign DNA,combating infection. This mechanism has similarities to RNA interferencefound in many eukaryotes. Because of its capabilities, Cas9 has beenused recently in experiments to serve as a genome editing tool.

CRISPR/Cas9 systems are a versatile tool for genome editing due to thehighly efficient targeting of DNA sequences complementary to their RNAguide strands. However, it has been shown that RNA guided Cas9 nucleasecleaves genomic DNA sequences containing mismatches to the guide strand.The use of chemically modified and protected nucleoside phosphoramiditesfor the synthesis of single guide RNAs (sgRNAs) to enhance genomeediting efficiency in human primary T cells, CD34+ hematopoietic stemand progenitor cells has been reported. The researchers argued thatco-delivery of chemically modified sgRNAs with Cas9 mRNA or protein isan efficient RNA- or ribonucleoprotein (RNP)-based delivery method forthe CRISPR-Cas system. This approach is a simple and effective way forthe development of new genome editing methods.

Additionally, genome editing with engineered nucleases is a breakthroughtechnology for modifying essentially any genomic sequence of interest.This technology exploits engineered nucleases to generate site-specificdouble-strand breaks (DSBs) followed by resolution of DSBs by endogenouscellular repair mechanisms. The outcome can be either mutation of aspecific site through mutagenic nonhomologous end-joining (NHEJ),creating insertions or deletions (in/dels) at the site of the break, orprecise change of a genomic sequence through homologous recombination(HR) using an exogenously introduced donor template. A recent majoraddition to this platform is the clustered regularly interspacedpalindromic repeat (CRISPR)/Cas system consisting of an RNA-guidednuclease (Cas) and a short guide RNA (sgRNA). The guide RNA is composedof two RNAs termed CRISPR RNA (crRNA) and trans-activating crRNA(tracrRNA), which are typically fused in a chimeric single guide RNA(sgRNA). sgRNAs for genome editing can consist of 100 nucleotides (nt)of which 20 nt at the 5′ end hybridize to a target DNA sequence by meansof Watson-Crick base pairing and guide the Cas endonuclease to cleavethe target genomic DNA.

The native prokaryotic CRISPR-Cas system comprises an array of shortrepeats with intervening variable sequences of constant length (i.e.,clusters of regularly interspaced short palindromic repeats, or“CRISPR”), and CRISPR-associated (“Cas”) proteins. The RNA of thetranscribed CRISPR array is processed by a subset of the Cas proteinsinto small guide RNAs, which generally have two components as discussedbelow. There are at least three different systems: Type I, Type II andType III. The enzymes involved in the processing of the RNA into maturecrRNA are different in the 3 systems. In the native prokaryotic system,the guide RNA (“gRNA”) comprises two short, non-coding RNA speciesreferred to as CRISPR RNA (“crRNA”) and trans-acting RNA (“tracrRNA”).In an exemplary system, the gRNA forms a complex with a Cas nuclease.The gRNA:Cas nuclease complex binds a target polynucleotide sequencehaving a protospacer adjacent motif (“PAM”) and a protospacer, which isa sequence complementary to a portion of the gRNA. The recognition andbinding of the target polynucleotide by the gRNA:Cas nuclease complexinduces cleavage of the target polynucleotide. The native CRISPR-Cassystem functions as an immune system in prokaryotes, where gRNA:Casnuclease complexes recognize and silence exogenous genetic elements in amanner analogous to RNAi in eukaryotic organisms, thereby conferringresistance to exogenous genetic elements such as plasmids and phages.

It has been demonstrated that a single-guide RNA (“sgRNA”) can replacethe complex formed between the naturally-existing crRNA and tracrRNA.

Considerations relevant to developing a gRNA, including a sgRNA, includespecificity, stability, and functionality. Specificity refers to theability of a particular gRNA:Cas nuclease complex to bind to and/orcleave a desired target sequence, whereas little or no binding and/orcleavage of polynucleotides different in sequence and/or location fromthe desired target occurs. Thus, specificity refers to minimizingoff-target effects of the gRNA:Cas nuclease complex. Stability refers tothe ability of the gRNA to resist degradation by enzymes, such asnucleases, and other substances that exist in intra-cellular andextra-cellular environments. Thus, there is a need for providing gRNA,including sgRNA, having increased resistance to nucleolytic degradation,increased binding affinity for the target polynucleotide, and/or reducedoff-target effects while, nonetheless, having gRNA functionality.Further considerations relevant to developing a gRNA includetransfectability and immunostimulatory properties. Thus, there is a needfor providing gRNA, including sgRNA, having efficient and titratabletransfectability into cells, especially into the nuclei of eukaryoticcells, and having minimal or no immunostimulatory properties in thetransfected cells. Another important consideration for gRNA is toprovide an effective means for delivering it into and maintaining it inthe intended cell, tissue, bodily fluid or organism for a durationsufficient to allow the desired gRNA functionality.

The CRISPR/Cas system has also been adapted for sequence-specificcontrol of gene expression, e.g., inhibition or activation of geneexpression. Using particular Cas9 polypeptide variants that lackendonuclease activity, target genes can be repressed or activated.Unfortunately, genome editing and modulating gene expression using theCRISPR/Cas system remains inefficient, especially in primary cells. Assuch, there remains a need in the art for improved compositions andmethods based on the CRISPR/Cas system that can be used for generegulation, e.g., genome editing with enhanced efficiency, inhibitinggene expression, and activating gene expression. The present inventionsatisfies this need and provides additional advantages as well.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the method of the invention using the XNA-RNA Chimerain CRISPR-Cas9 Gene Editing.

FIG. 2 features the sequences used for the gene editing using thexenonucleic acids of the invention.

FIG. 3 shows the efficiency of CRISPR mediated gene as measured by EGFPexpression in HEK293 cells.

FIGS. 4 and 5 illustrate the synthetic scheme for making theXNA-(linker)-crRNA.

SUMMARY OF THE INVENTION

The invention provides specific synthetic chimeric xenonucleic acidguide RNA; s(XNA-gRNA) for enhancing crispr mediated genome editingefficiency.

The present invention also provides a more efficient approach thatutilizes a chimeric XNA-gRNA construct that can be targeted specificallyto cells of interest. Since XNA's have a neutral non-phosphatecontaining backbone they are totally resistant to nucleases and alsobind more avidly to complementary target sequences that the natural RNAanalogs. XNA-gRNA chimeras (XNA-linker-RNA in FIG. 1.) can be targetedefficiently to any genetic locus and induce highly efficient CRISPRmediated gene editing at the targeted locus. The gene target sitespecific XNA-gRNA chimera is used together with a chemically synthesizedtrans-activating CRISPR RNA (tracrRNA) which complexes with the crRNA torecruit Cas9 nuclease.

The present invention also provides methods for inducing (e.g.,initiating, modulating, enhancing, etc.) gene editing/regulation of atarget nucleic acid in a cell. The invention includes using xenonucleicacid modified single guide RNAs (sgRNAs) that enhance genome editingand/or inhibition or activation of gene expression of a target nucleicacid in a primary cell (e.g., cultured in vitro for use in ex vivotherapy and other genetic engineering applications such as plantengineering) or in a cell in a subject such as a human (e.g., for use inin vivo therapy). The present invention also provides methods forpreventing or treating a disease in a subject by enhancing precisegenome editing to correct a mutation in a target gene associated withthe disease. The present invention can be used with any cell type and atany gene locus that is amenable to nuclease-mediated genome editingtechnology.

DETAILED DESCRIPTION OF THE INVENTION

The instant invention is directed to methods for enhancing theefficiency of CRISPR/Cas-based genome editing and/or modulation of geneexpression in an in vitro cell {e.g., a primary cell for use in ex vivotherapy) or an in vivo cell {e.g., a cell in an organ or tissue of asubject such as a human). The invention is applicable to any type ofcells i.e., cells derived from all mammals as well as other animalspecies and botanical plants. In particular, the methods provided hereinutilize single guide RNAs (sgRNAs) which have been modified withxenonucleic acids and therefore have enhanced activity during generegulation {e.g., genome editing, inhibition of gene expression, andactivation of gene expression) compared to corresponding unmodifiedsgRNAs. In certain aspects, the present invention provides methods forgene regulation of a target nucleic acid in a cell by introducing achemically modified sgRNA (i.e., a xenonucleic acid modified sgRNA) thathybridizes to the target nucleic acid together with either a Casnuclease {e.g., Cas9 polypeptide) or a variant or fragment thereof, anmRNA encoding a Cas nuclease {e.g., Cas9 polypeptide) or a variant orfragment thereof, or a recombinant expression vector comprising anucleotide sequence encoding a Cas nuclease {e.g., Cas9 polypeptide) ora variant or fragment thereof. In certain other aspects, the presentinvention provides methods for preventing or treating a genetic diseasein a subject by administering a sufficient amount of the xenonucleicacid modified sgRNA to correct a genetic mutation associated with thedisease.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of immunology, biochemistry,chemistry, synthetic organic chemistry, molecular biology, microbiology,cell biology, genomics and recombinant DNA, which are within the skillof the art. See Sambrook, Fritsch and Maniatis, Molecular Cloning: ALaboratory Manual, 2nd edition (1989), Current Protocols in MolecularBiology (F. M. Ausubel, et al. eds., (1987)), the series Methods inEnzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M. J.MacPherson, B. D. Hames and G. R. Taylor eds. (1995)), Harlow and Lane,eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (R.I. Freshney, ed. (1987)).

Oligonucleotides that are not commercially available can be chemicallysynthesized, e.g., according to the solid phase phosphoramidite triestermethod first described by Beaucage and Caruthers, Tetrahedron Lett. 22:1859-1862 (1981), using an automated synthesizer, as described in VanDevanter et. al, Nucleic Acids Res. 12:6159-6168 (1984). Purification ofoligonucleotides is performed using any art-recognized strategy, e.g.,native acrylamide gel electrophoresis or anion-exchange high performanceliquid chromatography (HPLC) as described in Pearson and Reanier, J.Chrom. 255: 137-149 (1983).

Unless specifically indicated otherwise, all technical and scientificterms used herein have the same meaning as commonly understood by thoseof ordinary skill in the art to which this invention belongs. Inaddition, any method or material similar or equivalent to a method ormaterial described herein can be used in the practice of the presentinvention.

The term “primary cell” refers to a cell isolated directly from amulticellular organism. Primary cells typically have undergone very fewpopulation doublings and are therefore more representative of the mainfunctional component of the tissue from which they are derived incomparison to continuous (tumor or artificially immortalized) celllines.

The term “genome editing” refers to a type of genetic engineering inwhich DNA is inserted, replaced, or removed from a target DNA, e.g., thegenome of a cell, using one or more nucleases and/or nickases. Thenucleases create specific double-strand breaks (DSBs) at desiredlocations in the genome, and harness the cell's endogenous mechanisms torepair the induced break by homology-directed repair (HDR) (e.g.,homologous recombination) or by nonhomologous end joining (NHEJ). Thenickases create specific single-strand breaks at desired locations inthe genome. In one non-limiting example, two nickases can be used tocreate two single-strand breaks on opposite strands of a target DNA,thereby generating a blunt or a sticky end. Any suitable nuclease can beintroduced into a cell to induce genome editing of a target DNA sequenceincluding, but not limited to, CRISPR-associated protein (Cas)nucleases, zinc finger nucleases (ZFNs), transcription activator-likeeffector nucleases (TALENs), meganucleases, other endo- orexo-nucleases, variants thereof, fragments thereof, and combinationsthereof. In particular embodiments, nuclease-mediated genome editing ofa target DNA sequence can be “induced” or “modulated” (e.g., enhanced)using the modified single guide RNAs (sgRNAs) described herein incombination with Cas nucleases (e.g., Cas9 polypeptides or Cas9 mRNA),e.g., to improve the efficiency of precise genome editing viahomology-directed repair (HDR).

The term “nucleic acid,” “nucleotide,” or “polynucleotide” refers todeoxyribonucleic acids (DNA), ribonucleic acids (RNA) and polymersthereof in either single-, double- or multi-stranded form. The termincludes, but is not limited to, single-, double- or multi-stranded DNAor RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprisingpurine and/or pyrimidine bases or other natural, chemically modified,biochemically modified, non-natural, synthetic or derivatized nucleotidebases. In some embodiments, a nucleic acid can comprise a mixture ofDNA, RNA and analogs thereof. Unless specifically limited, the termencompasses nucleic acids containing known analogs of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions), alleles,orthologs, single nucleotide polymorphisms (SNPs), and complementarysequences as well as the sequence explicitly indicated. Specifically,degenerate codon substitutions may be achieved by generating sequencesin which the third position of one or more selected (or all) codons issubstituted with mixed-base and/or deoxyinosine residues (Batzer et al,Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al, J. Biol. Chem.260:2605-2608 (1985); and Rossolini et al, Mol. Cell. Probes 8:91-98(1994)). The term nucleic acid is used interchangeably with gene, cDNA,and mRNA encoded by a gene.

The term “gene” or “nucleotide sequence encoding a polypeptide” meansthe segment of DNA involved in producing a polypeptide chain. The DNAsegment may include regions preceding and following the coding region(leader and trailer) involved in the transcription/translation of thegene product and the regulation of the transcription/translation, aswell as intervening sequences (introns) between individual codingsegments (exons). [0045] The terms “polypeptide,” “peptide,” and“protein” are used interchangeably herein to refer to a polymer of aminoacid residues. The terms apply to amino acid polymers in which one ormore amino acid residue is an artificial chemical mimetic of acorresponding naturally occurring amino acid, as well as to naturallyoccurring amino acid polymers and non-naturally occurring amino acidpolymers. As used herein, the terms encompass amino acid chains of anylength, including full-length proteins, wherein the amino acid residuesare linked by covalent peptide bonds.

The term “effective amount” or “sufficient amount” refers to the amountof an agent (e.g., Cas nuclease, modified single guide RNA, etc.) thatis sufficient to effect beneficial or desired results. Thetherapeutically effective amount may vary depending upon one or more of:the subject and disease condition being treated, the weight and age ofthe subject, the severity of the disease condition, the manner ofadministration and the like, which can readily be determined by one ofordinary skill in the art. The specific amount may vary depending on oneor more of: the particular agent chosen, the target cell type, thelocation of the target cell in the subject, the dosing regimen to befollowed, whether it is administered in combination with other agents,timing of administration, and the physical delivery system in which itis carried. [0058] The term “pharmaceutically acceptable carrier” refersto a substance that aids the administration of an agent (e.g., Casnuclease, modified single guide RNA, etc.) to a cell, an organism, or asubject. “Pharmaceutically acceptable carrier” refers to a carrier orexcipient that can be included in a composition or formulation and thatcauses no significant adverse toxicological effect on the patient.Non-limiting examples of pharmaceutically acceptable carrier includewater, NaCl, normal saline solutions, lactated Ringer's, normal sucrose,normal glucose, binders, fillers, disintegrants, lubricants, coatings,sweeteners, flavors and colors, and the like. One of skill in the artwill recognize that other pharmaceutical carriers are useful in thepresent invention. [0059] The term “increasing stability,” with respectto components of the CRISPR system, refers to modifications thatstabilize the structure of any molecular component of the CRISPR system.The term includes modifications that decrease, inhibit, diminish, orreduce the degradation of any molecular component of the CRISPR system.

The term “enhanced activity,” with respect to components of the CRISPRsystem and in the context of gene regulation, refers to an increase orimprovement in the efficiency and/or the frequency of inducing,modulating, regulating, or controlling genome editing and/or geneexpression.

The present invention provides methods for inducing gene regulation of atarget nucleic acid in a cell. The invention includes using xenonucleicacid modified single guide RNAs (sgRNAs) that enhance genome editingand/or inhibition or activation of gene expression of a target nucleicacid in a primary cell (e.g., cultured in vitro for use in ex vivotherapy) or in a cell in a subject such as a human (e.g., for use in invivo therapy). The present invention also provides methods forpreventing or treating a disease in a subject by enhancing precisegenome editing to correct a mutation in a target gene associated withthe disease. The present invention can be used with any cell type and atany gene locus that is amenable to nuclease-mediated genome editingtechnology.

The present invention is also directed to a method for inducing (e.g.,initiating, modulating, enhancing, efficient editing, etc.) generegulation of a target nucleic acid in a primary cell, the methodcomprising: introducing into the primary cell: (a) a xenonucleic acidmodified single guide RNA (sgRNA) comprising a first nucleotide sequencethat is complementary to the target nucleic acid and a second nucleotidesequence that interacts with a CRISPR-associated protein (Cas)polypeptide, wherein one or more of the nucleotides in the firstnucleotide sequence and/or the second nucleotide sequence arenucleotides which have been chemically modified with xenonucleic acids;and (b) a Cas polypeptide, an mRNA encoding a Cas polypeptide, and/or arecombinant expression vector comprising a nucleotide sequence encodinga Cas polypeptide, wherein the xenonucleic acid modified sgRNA guidesthe Cas polypeptide to the target nucleic acid, and wherein thexenonucleic acid modified sgRNA induces gene regulation of the targetnucleic acid with an enhanced activity and efficiency relative to acorresponding unmodified sgRNA.

The enhanced activity and efficiency comprises increased stability ofthe xenonucleic acid modified sgRNA and/or increased specificity of themodified sgRNA for the target nucleic acid. In some embodiments, thetarget nucleic acid comprises a target DNA or a target RNA. Generegulation of a target nucleic acid encompasses any mechanism used bycells to increase or decrease the production of a specific gene product(e.g., protein or RNA) by the target nucleic acid and includes efficientgenome editing of the target nucleic acid or modulation (e.g.,inhibition or activation) of gene expression of the target nucleic acid.In some instances, the gene regulation comprises genome editing of thetarget DNA. The genome editing can be homologous-directed repair (HDR)or nonhomologous end joining (HEJ) of the target DNA. In other cases,the gene regulation comprises modulating (e.g., inhibiting oractivating) gene expression of the target DNA or target RNA using anendonuclease-deficient Cas polypeptide.

The Xenonucleic acids used in the invention are new nucleic acidmolecular oligomers that hybridize by Watson-Crick base pairing totarget DNA sequences yet have a modified chemical backbone. Thexenonucleic acid oligomers are highly effective at hybridizing to targetsequences and can be employed as molecular clamps in quantitativereal-time polymerase chain reactions or as highly specific molecularprobes for detection of nucleic acid target sequences.

This invention is also based, at least in part, on an unexpecteddiscovery that certain chemical modifications to gRNA are tolerated bythe CRISPR-Cas system. In particular, certain chemical modificationsbelieved to increase the stability of the gRNA, to alter thethermostability of a gRNA hybridization interaction, and/or to decreasethe off-target effects of Cas:gRNA complexation do not substantiallycompromise the efficacy of Cas:gRNA binding to, nicking of, and/orcleavage of the target polynucleotide. Furthermore, certain chemicalmodifications are believed to provide gRNA, including sgRNA, havingefficient and titratable transfectability into cells, especially intothe nuclei of eukaryotic cells, and/or having minimal or noimmunostimulatory properties in the transfected cells. Certain chemicalmodifications are believed to provide gRNA, including sgRNA, which canbe effectively delivered into and maintained in the intended cell,tissue, bodily fluid or organism for a duration sufficient to allow thedesired gRNA functionality.

For purposes of illustration, the scheme below illustrates thedifferences between DNA and XNA:

Applicant has developed a multitude of XNA chemistry and multipleapplications of XNA in molecular testing including, PCR-Clamping,in-situ detection of gene mutations and targeted CRISPR/Cas9gene-editing and detection. Applicant's XNA chemistry is unique in thata single nucleotide change in the target sequence can lead to a meltingtemperature differential of as much as 15-200 C. For natural DNA the Tmdifferential for such a change is only 5-70 C.

Representative examples are shown below:

The XNA monomers are synthesized as shown in the following schemes:

Synthesis of Xenonucleic Acid (XNA) Monomers

Aza-XNA Monomer Synthesis

Synthesis of Oxaza-XNA Monomer

Attachment of Protected Nucleic Acid Bases and Solid Phase Synthesis ofXNA Oligomers

Benzothiazole-2-Sulfonyl-(Bts) Route to XNA Monomer Synthesis

We could also introduce CDI (carbonyldiimidazole chemistry; by doingthat we may skip Step 7 in above and can get to the final cyclizedmonomer.

The azide derivatized XNA is made via azidobutyrate NHS ester can beused to introduce an active azide group to an amino-modifiedoligonucleotide. Introduction can be done at either the 5′- or 3′-end,or internally. To do this, the oligo first must be synthesized with aprimary amino functional group modification, e.g. amino C₆ for the 5′end or amino C₇ for the 3′ end for the ends) or the amino C₆ version ofthe base phosphoramidite (for internal labeling). The Azidobutyrate NHSester is then manually attached to the oligo through the amino group ina separate reaction post-synthesis. The presence of the azide allows theuser to use “Click Chemistry” (a [3+2] cycloaddition reaction betweenalkynes and azides, using copper (I) iodide as a catalyst) to conjugatethe azide-modified oligo to a terminal alkyne-modified oligo withextremely high regioselectivity and efficiency.

A representative chemical structure is as follows:

In one embodiment, the XNA-gRNA chimera are synthesized by chemicalcoupling of 3′-modified XNA oligomer with a suitable 5′-modifiedsynthetic RNA oligomer using conjugation chemistries that are well knownin the art. An example as mentioned above is “Click chemistry” utilizingalkynyl modified linkers and/or nucleosides and azide modified linkersfor attachment.

Click chemistry involves the rapid generation of compounds by joiningsmall units together via heteroatom links (C-X-C). The main objective ofclick chemistry is to develop a set of powerful, selective, and modular“blocks” that are useful for small- and large-scale applications.Reaction processes involved in click chemistry should conform to adefined set of stringent criteria such as being: Simple to perform,modular, wide in scope, high yielding, stereospecific, environmentallyfriendly by generating only harmless byproducts that can be removed bynon-chromatographic methods.

Important characteristics of the reactions involved in click chemistryare: simple reaction conditions, readily and easily available startingmaterials and reagents, use of no solvent, a benign solvent (such aswater), or one that is easily removed, simple product isolation andproduct should be stable under physiological conditions.

Click chemistry involves the use of a modular approach and has importantapplications in the field of drug discovery, combinatorial chemistry,target-templated in situ chemistry, and DNA research.

A well-known click reaction is the Huisgen 1,3-dipolar cycloaddition ofazides and alkynes. This reaction, yielding triazoles, has become thegold standard of click chemistry for its reliability, specificity, andbiocompatibility. Such cycloadditions need high temperatures orpressures when the reaction involves simpler alkene or azides, since theactivation energies are high (ΔG^(□)≈+26 kcal/mol). Sharpless &co-workers and Meldal & co-workers reported Cu(I) catalysts expedite thereaction of terminal alkynes and azides, thereby affording1,4-disubstituted triazoles. This reaction is an ideal click reactionand is widely employed in material science, medicinal chemistry, andchemical biology.

The Scheme of the well-known Cu-catalyzed azide-alkyne cycloadditionreaction:

The cytotoxic nature of transition metals, employed as catalysts for theclick reactions, precluded their use for in vivo applications.Alternative approaches with lower activation barriers and copper-freereactions were proposed. Such reactions were referred to as “copper-freeclick chemistry”. Copper-free click chemistry is based on a very oldreaction, published in 1961 by Wittig et al. It involved the reactionbetween cyclooctyne and phenyl azide, which proceeded like an explosionto give a single product,1-phenyl-4,5,6,7,8,9-hexahydro-1H-cycloocta[d][1,2,3]triazole. Thereaction is ultrafast due to the large amount of ring-strain (18kcal/mol of ring strain) in the cyclooctyne molecule. Release of thering-strain in the molecule drives the fast reaction. Cyclooctynes arereported to react selectively with azides to form regioisomeric mixturesof triazoles at ambient temperatures and pressures without the need formetal catalysis and no apparent cytotoxicity. Difluorinated cyclooctynereagents have been reported to be useful for the copper-free clickchemistry.

Co-delivering chemically modified sXNA-gRNAs with Cas9 mRNA or proteinis an efficient RNA- or ribonucleoprotein (RNP)-based delivery methodfor the CRISPR-Cas system, without the toxicity associated with DNAdelivery. This approach is a simple and effective way to streamline thedevelopment of genome editing with the potential to accelerate a widearray of biotechnological and therapeutic applications of the CRISPR-Castechnology.

Very little is known about the tolerance of the gRNAs of Cas9 and Cpf1towards chemical modifications. Without this information, it ischallenging to rationally engineer gRNAs for biotechnologicalapplications. Also ‘off-target’ binding of crRNA's is a problem forspecificity of targeted NHEJ or HDR mediated editing.

Thus we generated chemically modified CRISPR targeting RNAs (crRNAs),which had XNA or donor DNA sequence(s) attached at their 5′ or 3′ ends,and evaluated their ability to cleave genomic DNA, after complexationwith Cas9, in cells expressing green fluorescent protein (GFP) undercontrol of the TET on/off promoter system. The constructs consisted ofcrRNAs targeting the GFP sequence, which had a short single stranded XNA(15-24 nucleobases) or donor DNA (82-87 nucleotides), at their 5′ or 3′position. These modifications were chosen because of their importance inperforming conjugation reactions.

Exemplary synthesis of 5′-XNA linked crRNA is shown below:

The linker length that is used in the conjugate is determinedempirically based on the target binding sequence that is distal (i.e.5′-upstream of 3′ downstream of the CRISPR edit site in the targetgene.)

We selected as a target gene to demonstrate the utility of our approachthe tetracycline inducible EGFP reporter (TET on/off) system in HEK293cells. CRISPR gRNA was targeted to inactivate the TET repressor.Efficient generation of deletions in this target region would lead toexpression of the EGFP reporter gene which can be measures byfluorescence microscopy and/or FACS analysis

For TET repressor EGFP reporter targeted CRISPR/Cas9 mediated geneediting the sequence of the crRNA and tracrRNA is shown below:

CRISPR gRNA: SEQ ID NO: 1 5′-gUGGACUCAUGAUCACGGGUCGUUUUAGAGCUA-3′tracrRNA: SEQ ID NO: 2 5′-AGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGC-3′ TET Repressor EGFP/CRISPR/Cas9 Target Site:SEQ ID NO: 3                    PAM   I Cut Site AGATCTACCATGCCAAAGAGACCCA GACCCGTGATCATGAGTCCAAAGA GAAGAACACAGGCAGAGCGCGCAATGGAGACCCAGSEQ ID NO: 4 TCTAGATGGTACGGTTTCTCTG GGT CTGGGCACTAGTACTCAGGTTTCTCTTCTTGTGTCCGTCTCGCGCGTTACCTCTGGGTC                                   gRNACRISPR/Cas9 disruption of the TET repressor leads to inducibleexpression of EGFP reporter in HEK293 cells. The % modification ismeasured employing detection of EGFP expression in the presence oftetracycline. High EGFP expression implies efficient KO of the TETrepressor by CRISPR/Cas9. FIG. 2 shows that synthetic sXNA-crRNA is muchmore efficient than scrRNA alone and radically more efficient thatplasmid derived crRNA.

Additional CRISPR gene-editing target xeno-clamp sequences that can beused in the present invention include:

GBP1 edit site clamp, SEQ ID NO: 5 D-LYS-O-AGAGTTGTGTCGTCGA;Wild-type xeno-clamp for a target gene, SEQ ID NO: 6D-LYS-O-TTTCTACGCTCAGCCTT GG;Mutant specific xeno-clamp for a target gene, SEQ ID NO: 7D-LYS-O-TTTCTCCGCTCAGCC TTGG

Clamp (1) is for a gene: GBP1 that is responsible for development ofresistance to therapy in ovarian cancer. Clamps (2) and (3) are designedto be used when the target gene to be edited is a heterozygote i.e. thetarget site has a heterozygous mutation in the vicinity of the CRISPRedit site! So it is very difficult to determine editing efficiency sincethe target gene already has an endonuclease cleavage site present evenbefore CRISPR editing. Using wild-type and mutant specific clamps is theonly way to determine editing efficiency.

Other xenoclamps include:

WTAP CRISPR target (NEB), SEQ ID NO: 8 AcACCCACAGTTCGATT-NH₂ and GFPgene editing site XNA clamp sequence, SEQ ID NO: 95′-D-LYS-O-CCGGTCAGCTCG AT-3′.

Additional xenoclamps that can be used in the invention include oxy-azaand aza XNAs described in the table below.

Sequence Name Oxy-Aza Aza XNA BR001 SEQ ID NO: 10ATCGAGATTTCACTGTAGCTAGAC x DPCA001 SEQ ID NO: 11 ACTTCAGGCAGCGTCTTCA xDPCA002 SEQ ID NO: 12 TGTTCAGAGCACACTTCAG x DPCA003 SEQ ID NO: 13CTGGTGGTTGAATTTGCTG x DPCA004 SEQ ID NO: 14 CATGAGCTCCAGCAGGATGAAC xDPCA005 SEQ ID NO: 15 CCGAAGTCTCCAATCTTGG x DPCA006 SEQ ID NO: 16TAGATGTCTCGGGCCATCC x DPCBRC001 SEQ ID NO: 17 GGGACACTCTAAGAT xDPCBRC002 SEQ ID NO: 18 TTCTGTCCTGGGATTCTC x DPCBRC003 SEQ ID NO: 19AGATTTTCCACTTGCTGT x DPCBRCA001-2 SEQ ID NO: 20CCAGATGGGACACTCTAAGATTTTC x DPCBRCA002-2 SEQ ID NO: 21CCTTTCTGTCCTGGGATTCTCTT x DPCBRCA003-2 SEQ ID NO: 22GACAGATTTTCCACTTGCTGTGCTAA x DPCBRCA004 SEQ ID NO: 23CATAAAGGACACTGTGAAGGCC x DPCBRCA004B SEQ ID NO: 24D-LYS-O-GGCCTTCACAGTGTCCTTTA TG x DPCCKT002 SEQ ID NO: 25D-LYS-O-CATTCTTGATGTCTCTGGCT AG x DPCE001 SEQ ID NO: 26 GAGCCCAGCACTTT xDPCE001B SEQ ID NO: 27 D-LYS-O-CGGAGCCCAGCACTTTGAT x DPCE001B1SEQ ID NO: 28 D-LYS-O-CGGAGCCCAGCACTTTGAT x DPCE002 SEQ ID NO: 29NH(2)-AGATGTTGCTTCTCTTAA-CONH(2) x DPCE002B SEQ ID NO: 30D-LYS-O-AGATGTTGCTTCTCTTAA x DPCE002C SEQ ID NO: 31D-LYS-O-CGGAGATGTTGCTTCTCTTAATTCC x DPCE004 SEQ ID NO: 32CAGTTTGGCCAGCCCA x DPCE004B SEQ ID NO: 33 CAGTTTGGCCAGCCCA-O-D-LYS xDPCE004C SEQ ID NO: 34 D-LYS-O-TTTGGCCAGCCCAAAATCTGT x DPCE004DSEQ ID NO: 35 D-LYS-O-GGCCAGCCCAAAATCTGT x DPCE005 SEQ ID NO: 36ACCCAGCAGTTTGGC x DPCE005B SEQ ID NO: 37 D-LYS-O-ACCCAGCAGTTTGGC xDPCE006 SEQ ID NO: 38 GCTGCGTGATGAG x DPCE007 SEQ ID NO: 39 GCTGCGTGATGAx DPCE008 SEQ ID NO: 40 AGCTCATCACGCAGCTCATG x DPCE008B SEQ ID NO: 41D-LYS-O-CAGCTCATCACGCAGCTCATGC x DPCE008C SEQ ID NO: 42D-LYS-O-TCATCACGCAGCTCATGCCCTT x DPCE008D SEQ ID NO: 43D-LYS-O-CTCATCACGCAGCTCATG x DPCE008E SEQ ID NO: 44D-LYS-O-TGAGCTGCGTGATG x DPCE009B SEQ ID NO: 45D-LYS-O-TCCACGCTGGCCATCACGTA x DPCE009B-1 SEQ ID NO: 46TCCACGCTGGCCATCACGTA-O-D-LYS x DPCE010B SEQ ID NO: 47TGGGGGTTGTCCAC-O-D-LYS x DPCE011 SEQ ID NO: 48 GCACACGTGGGGGTT-O-D-LYS xDPCE012 SEQ ID NO: 49 D-LYS-O-ACAACCCCCACGTGTGC x DPCH001 SEQ ID NO: 50CTGAGCCAGGAGAAAC x DPCH002 SEQ ID NO: 51 GTAAACTGAGCCAGGAG x DPCH003SEQ ID NO: 52 ATGGCACTAGTAAACTGAGC x DPCH004 SEQ ID NO: 53ATCCATATAACTGAAAGCCAA x DPCH005 SEQ ID NO: 54 ACCACATCATCCATATAACTGAA xDPCHRAS001B SEQ ID NO: 55 D-LYS-O-O-TTGCCCACACCGCCGGC x DPCHRAS002SEQ ID NO: 56 D-LYS-O-O-TCTTGCCCACACCGCC x DPCHRAS003 SEQ ID NO: 57D-LYS-O-O-TACTCCTCCTGGCCGGC x DPCJ001 SEQ ID NO: 58CGTCTCCACAGACACATACTCCA x DPCJ002B SEQ ID NO: 59CGTCTCCACAGACACATACTCCA-O-D-LYS x DPCK001B SEQ ID NO: 60GCCTACGCCACCAGCTCCAAC-O-D-LYS x DPCK001B2 SEQ ID NO: 61GCCTACGCCACCAGCTCCAAC-O-O-D-LYS x DPCK001C SEQ ID NO: 62CTACGCCACCAGCTCCAACTACCA x DPCK001C2 SEQ ID NO: 63CTACGCCACCAGCTCCAACTACCA-O-D-LYS x DPCK002 SEQ ID NO: 64TCTTGCCTACGCCACCAGCTCCA x DPCK003 SEQ ID NO: 65 TGTACTCCTCTTGACCTGCTGTGx DPCK003B SEQ ID NO: 66 D-LYS-O-TGTACTCCTCTTGACCTGCTGTG x DPCK004SEQ ID NO: 67 NH(2)-GGCAAATCACATTTATTTCCTAC-CONH(2) x DPCK004BSEQ ID NO: 68 D-LYS-O-GGCAAATCACATTTATTTCCTAC x DPCK005B SEQ ID NO: 69D-LYS-O-TGTCTTGTCTTTGCTGATGTTTC x DPCK005 SEQ ID NO: 70TGTCTTGTCTTTGCTGATGTTTC x DPCK005C SEQ ID NO: 71D-LYS-O-TGTCTTGTCTTTGCTGATGTTTC x DPCK006 SEQ ID NO: 72NH(2)-CTCTTGACCTGCTGTGTCGAG-CONH(2) x DPCN001 SEQ ID NO: 73TCCCAACACCACCTGCTCCAA x DPCN001B SEQ ID NO: 74D-LYS-O-CAACACCACCTGCTCCAACCACCAC x DPCN002 SEQ ID NO: 75CTTTTCCCAACACCACCTGCTCC x DPCN002B SEQ ID NO: 76D-LYS-O-TGCGCTTTTCCCAACACCACCTGCT x DPCN003B SEQ ID NO: 77GGCACTGTACTCTTCTTGTCCAG x DPCN004B SEQ ID NO: 78D-LYS-O-TCTGGTCTTGGCTGAGGTTTC x DPCN006 SEQ ID NO: 79NH(2)-GGCAAATCACACTTGTTTCCCAC-CONH(2) x DPCN006B SEQ ID NO: 80D-LYS-O-GGCAAATCACACTTGTTTCCCAC x DPCN007 SEQ ID NO: 81NH(2)-TTCTTGTCCAGCTGTATCCAGTATG-CONH(2) x DPCPKA003B SEQ ID NO: 82D-LYS-O-AGATCCTCTCTCTGAAATCAC x DPCPKA004 SEQ ID NO: 83D-LYS-O-TCTTTCTCCTGCTCAGTGATTTCA x DPCPKA005 SEQ ID NO: 84D-LYS-O-AATGATGCACATCATGGTGGCTG x NRASN003C SEQ ID NO: 85D-LYS-O-GGCACTGTACTCTTCTTGTCCAG x QMDXNA001 SEQ ID NO: 86NH(2)-O-TTCATCAACCGCACTCTGTTTATCTC x QMDXNA002 SEQ ID NO: 87NH(2)-O-TGGCGACGACAATGGACCCAATTAT x QMDXNA003 SEQ ID NO: 88NH(2)-O-AGATGTAGTTAGCAATCGGTCCTTGTTGTA x QMDXNA004 SEQ ID NO: 89NH(2)-O-GGGTAATTGAGGTAACGTAGGTATCAAGAT x QMDXNA005 SEQ ID NO: 90NH(2)-O-TACTATCGACTGACATGAGGCTTGTGT x XNADE001 SEQ ID NO: 91D-LYS-O-AGTCCGACGATCTGGAATTC x XNADE002 SEQ ID NO: 92D-LYS-O-ACTGGAGTTCAGACGTGTG x XNADE003 SEQ ID NO: 93D-LYS-O-CTCTTCCGATCAGATCGGAA x XNADE003b SEQ ID NO: 94D-LYS-O-CTCTTCCGATCAGATCGGAAG x XNAFGFR001 SEQ ID NO: 95D-LYS-O-O-AGCGCTCCCCGCACC x XNAFGFR001 SEQ ID NO: 96D-LYS-O-O-AGCGCTCCCCGCACC x XNAFGFR002 SEQ ID NO: 97D-LYS-O-GGGGAGCGCTCTGT-O-TTTTT x XNAFGFR003 SEQ ID NO: 98D-LYS-O-O-AGCGCTCCCCGCACC-O-TTTTTT x XNAFGFR004 SEQ ID NO: 99D-LYS-O-TGCATACACACTGCCCGCCT x

EXAMPLES

Click chemistry is a versatile reaction that can be used for thesynthesis of a variety of conjugates. Virtually any biomolecules can beinvolved, and labeling with small molecules, such as fluorescent dyes,biotin, and other groups can be readily achieved.

Click chemistry reaction takes place between two components: azide andalkyne (terminal acetylene). Both azido and alkyne groups are nearlynever encountered in natural biomolecules. Hence, the reaction is highlybioorthogonal and specific. If there is a need to label anoligonucleotide, alkyne-modified oligonucleotides can be ordered at manyof the custom oligo-synthesizing facilities and companies.We recommend using the following general protocol for Click chemistrylabeling of alkyne-modified oligonucleotides with azides produced byLumiprobe Corp. The auxiliary reagents can be ordered at Lumiprobe Corp.

1. Calculate the volumes of reagents required for Click chemistrylabeling using the table below. Prepare the required stock solutions.

Final concentration Stock solution Reagent in the mixture concentrationOligonucleotide, Varies (20-200 uM) varies alkyne-modified Azide 1.5 ×(oligonucleotide 10 mM in DMSO concentration) DMSO 50 vol % — Ascorbicacid 0.5 mM 5 mM in water Cu-TBTA complex 0.5 mM 10 mM in 55 vol % DMSO1. Dissolve alkyne-modified oligonucleotide or DNA in water in apressure-tight vial.2. Add 2M triethylammonium acetate buffer, pH 7.0, to finalconcentration 0.2 M.3. Add DMSO, and vortex.4. Add azide stock solution (10 mM in DMSO), and vortex.5. Add the required volume of 5 mM Ascorbic Acid Stock solution to themixture, and vortex briefly.6. Degass the solution by bubbling inert gas in it for 30 seconds.Nitrogen, argon, or helium can be used.7. Add the required amount of 10 mM Copper (II)-TBTA Stock in 55% DMSOto the mixture. Flush the vial with inert gas and close the cap.8. Vortex the mixture thoroughly. If significant precipitation of azideis observed, heat the vial for 3 minutes at 80° C., and vortex.9. Keep at room temperature overnight.10. Precipitate the conjugate with acetone (for oligonucleotides) orwith ethanol (for DNA). Add at least 4-fold volume of acetone to themixture (If the volume of the mixture is large, split in several vials).Mix thoroughly and keep at −20° C. for 20 minutes.11. Centrifuge at 10000 rpm for 10 minutes.12. Discard the supernatant.13. Wash the pellet with acetone (1 mL), centrifuge at 10000 rpm for 10minutes.14. Discard the supernatant, dry the pellet, and purify the conjugate byRP-HPLC or PAGE.

XNA-crRNA Synthesis and Purification

XNA(s) containing 3′-azide monomer were synthesized on a 5-μmol scale onan Applied Biosystems 433A peptide synthesizer. Resin used was NovaSynTGR (rink amide) resin preloaded with FMoc-D-lysine (substitution 0.045meq/g). 3′-azido-XNA (10 mM) was mixed with 5′-DBCO-crRNA (30 mM) in DIwater (50 mL). The solution was incubated at room temperature over-nightand the unreacted crRNA was removed by running the reaction solutionthrough a 30k concentrator (Amicon Ultra, EMD Millipore). The XNA-crRNAreaction solution was analyzed via gel electrophoresis using apolyacrylamide gel (4-20% Mini-protean TGX Precast gel, Biorad) 200 ngof the reaction mixture was loaded into the gel. The XNA-crRNA band wascut with a sharp knife and eluted using the crush and soak method innuclease-free water for 16 hr, and isolated via ethanol precipitation.

NanoFect™ Transfection Reagent (Alstem, Cat#NF100)

The following protocol was used for transfection in a 24-well plate.1. For each well, add 0.5 ml of normal growth medium (antibiotic doesnot influence the result) freshly2 hours before transfection.2. For each well, dilute 0.5 μg of DNA in 50 μl of DMEM without serum,and mix gently.3. Add 1.5 μl of NanoFect™ reagent (ALSTEM, Cat. #NF100) into anothertube with 50 μl of DMEMwithout serum, and mix gently.4. Add NanoFect™/DMEM into DNA/DMEM solution. Mix by vortexing for 5-10seconds.5. Incubate for ˜15 minutes at room temperature to allow forNanoFect™/DNA complexes self-assembly.6. Add the 100 μl NanoFect™/DNA mix drop-wise to the cells in each welland homogenize by gentlyswirling the plate.7. Return the plates to the cell culture incubator.8. Check transfection efficiency under fluorescent microscopy or FACSsorting cells 24 to 48 hours post transfection.

REFERENCES

-   1) Chemically modified guide RNAs enhance CRISPR-Cas genome editing    in human primary cells. A. Hendel et. al., Nature Biotechnology    2015, 33, 985-989 DOI: 10.1038/nbt.3290-   2) Site-specific terminal and internal labeling of RNA by poly(A)    polymerase tailing and copper-catalyzed or copper-free    strain-promoted click chemistry. M.-L. Winz et. al., Nucleic Acids    Research, 2012, 1-13 DOI:10.1093/nar/gks062-   3) A. V. Ustinov, I. A. Stepanova, V. V. Dubnyakova, T. S.    Zatsepin, E. V. Nozhevnikova, V. A. Korshun. Modification of nucleic    acids using [3+2]-dipolar cycloaddition of azides and alkynes.    Russ. J. Bioorg. Chem. 36(4), 401-445 (2010). DOI:    10.1134/S1068162010040011-   4) A. H. El-Sagheer, T. Brown. Click chemistry with DNA. Chem. Soc.    Rev. 39(4), 1388-1405 (2010). DOI: 10.1039/B901971P-   5) F. Amblard, J. H. Cho, R. F. Schinazi. Cu(I)-catalyzed Huisgen    azide-alkyne 1,3-dipolar cycloaddition reaction in nucleoside,    nucleotide, and oligonucleotide chemistry. Chem. Rev. 109(9),    4207-4220 (2009). DOI: 10.1021/cr9001462-   6) F. Zhang et. al., Genome engineering using the CRISPR-Cas9 system    Nature Protocols, 2013, 8 (11), 2281-2308

The content of all references cited in the instant specification and allcited references in each of those references are incorporated in theirentirety by reference herein as if those references were denoted in thetext

While the many embodiments of the invention have been disclosed (Angres)above and include presently preferred embodiments, many otherembodiments and variations are possible within the scope of the presentdisclosure and in the appended claims that follow. Accordingly, thedetails of the preferred embodiments and examples provided are not to beconstrued as limiting. It is to be understood that the terms used hereinare merely descriptive rather than limiting and that various changes,numerous equivalents may be made without departing from the spirit orscope of the claimed invention.

What is claimed is:
 1. RNA modified with a linker and a xenonucleic acid wherein said xenonucleic acid has functionality selected from the group consisting of azide, oxaaza and aza.
 2. The modified RNA of claim 1, wherein said RNA is sgRNA.
 3. The modified RNA of claim 1, wherein said RNA is crRNA.
 4. The modified RNA of claim 1, wherein said RNA is tracrRNA.
 5. The modified RNA of claim 1, having the formula:


6. The modified RNA of claim 1, wherein said RNA is chimeric.
 7. A method for inducing gene regulation of a target nucleic acid in a primary cell, the method comprising: introducing into the primary cell: (a) a xenonucleic acid modified single guide RNA (sgRNA) comprising a first nucleotide sequence that is complementary to the target nucleic acid and a second nucleotide sequence that interacts with a CRISPR-associated protein (Cas) polypeptide, wherein one or more of the nucleotides in the first nucleotide sequence and/or the second nucleotide sequence are nucleotides which have been chemically modified with xenonucleic acids; and (b) a Cas polypeptide, an mRNA encoding a Cas polypeptide, and/or a recombinant expression vector comprising a nucleotide sequence encoding a Cas polypeptide, wherein the xenonucleic acid modified sgRNA guides the Cas polypeptide to the target nucleic acid, and wherein the xenonucleic acid modified sgRNA induces gene regulation of the target nucleic acid with an enhanced activity and efficiency relative to a corresponding unmodified sgRNA.
 8. A method for preventing or treating a genetic disease in a subject or a plant, the method comprising: administering to the subject or plant a xenonucleic acid modified single guide RNA (sgRNA) in a sufficient amount to correct a mutation in a target gene associated with the genetic disease, wherein the xenonucleic acid modified sgRNA comprises a first nucleotide sequence that is complementary to the target gene and a second nucleotide sequence that interacts with a CRISPR-associated protein (Cas) polypeptide, and wherein one or more of the nucleotides in the first nucleotide sequence and/or the second nucleotide sequence are xenonucleic acid modified nucleotides. 